ABSTRACT
OBJECTIVES: The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures. METHODS: An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. RESULTS: Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. CONCLUSIONS: Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.
Subject(s)
Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Influenza A virus/drug effects , Influenza A virus/physiology , RNA Cap-Binding Proteins/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Virus Replication/drug effects , Animals , Antiviral Agents/toxicity , Cell Line , Cell Survival/drug effects , Disease Models, Animal , Drug Synergism , Female , Fluorescence Polarization , Humans , Mice, Inbred BALB C , Orthomyxoviridae Infections/drug therapy , Treatment OutcomeABSTRACT
Antivirals are used not only in the current treatment of influenza but are also stockpiled as a first line of defense against novel influenza strains for which vaccines have yet to be developed. Identifying drug resistance mutations can guide the clinical deployment of the antiviral and can additionally define the mechanisms of drug action and drug resistance. Pimodivir is a first-in-class inhibitor of the polymerase basic protein 2 (PB2) subunit of the influenza A virus polymerase complex. A number of resistance mutations have previously been identified in treated patients or cell culture. Here, we generate a complete map of the effect of all single-amino-acid mutations to an avian PB2 on resistance to pimodivir. We identified both known and novel resistance mutations not only in the previously implicated cap-binding and mid-link domains, but also in the N-terminal domain. Our complete map of pimodivir resistance thus enables the evaluation of whether new viral strains contain mutations that will confer pimodivir resistance.
Subject(s)
Antiviral Agents/pharmacology , Birds/virology , Drug Resistance, Viral/genetics , Influenza A virus/genetics , Mutation , Pyridines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Viral Proteins/genetics , A549 Cells , Animals , Genetic Variation , Humans , Influenza A virus/classification , Influenza in Birds/virology , RNA Cap-Binding Proteins/antagonists & inhibitors , Viral Proteins/chemistryABSTRACT
BACKGROUND: Hypoxia suppresses global protein production, yet certain essential proteins are translated through alternative pathways to survive under hypoxic stress. Translation via the internal ribosome entry site (IRES) is a means to produce proteins under stress conditions such as hypoxia; however, the underlying mechanism remains largely uncharacterized. METHODS: Proteomic and bioinformatic analyses were employed to identify hnRNPM as an IRES interacting factor. Clinical specimens and mouse model of tumorigenesis were used for determining the expression and correlation of hnRNPM and its target gene. Transcriptomic and translatomic analyses were performed to profile target genes regulated by hnRNPM. FINDINGS: Hypoxia increases cytosolic hnRNPM binding onto its target mRNAs and promotes translation initiation. Clinical colon cancer specimens and mouse carcinogenesis model showed that hnRNPM is elevated during the development of colorectal cancer, and is associated with poor prognosis. Genome-wide transcriptomics and translatomics analyses revealed a unique set of hnRNPM-targeted genes involved in metabolic processes and cancer neoplasia are selectively translated under hypoxia. INTERPRETATION: These data highlight the critical role of hnRNPM-IRES-mediated translation in transforming hypoxia-induced proteome toward malignancy. FUND: This work was supported by the Ministry of Science and Technology, Taiwan (MOST 104-2320-B-006-042 to HSS and MOST 105-2628-B-001-MY3 to TMC).
Subject(s)
Cell Hypoxia , Colonic Neoplasms/pathology , Heterogeneous-Nuclear Ribonucleoprotein Group M/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Cluster Analysis , Colonic Neoplasms/chemically induced , Colonic Neoplasms/mortality , Disease Models, Animal , Eukaryotic Initiation Factor-4E , Fibroblast Growth Factor 9/genetics , Fibroblast Growth Factor 9/metabolism , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoprotein Group M/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein Group M/genetics , Humans , Kaplan-Meier Estimate , Mice , Protein Biosynthesis , RNA Cap-Binding Proteins/antagonists & inhibitors , RNA Cap-Binding Proteins/genetics , RNA Cap-Binding Proteins/metabolism , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
Mevalonate biosynthesis pathway is important in cell growth and survival and its blockade by 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, arrest brain neuroblasts growth and induce apoptosis. Translation is among the main biochemical mechanisms that controls gene expression and therefore cell growth or apoptosis. In the CNS, translation regulates synaptic plasticity. Thus, our aim was to investigate the effect of lovastatin in protein translation in rat neuroblasts of the CNS and the biochemical pathways involved. Lovastatin treatment in rat brain neuroblasts causes a significant time- and concentration-inhibition of protein synthesis, which is partially mediated by phosphatydilinositol 3-kinase/mammalian target of rapamycin (mTOR) pathway inhibition. Lovastatin treatment decreases the phosphorylation state of mTOR substrates, p70S6K and eukaryotic translation initiation factor (eIF) 4E-binding protein 1 and simultaneously increases eIF4E-binding protein 1 in a time-dependent manner. Concomitantly, lovastatin causes a decrease in eIF4G cellular amount, which is partially mediated by caspase(s) activity excluding caspase 3. These biochemical pathways affected by lovastatin might explain the protein translation inhibition observed in neuroblasts. Cycloheximide treatment, which blocked protein synthesis, does not induce neuroblasts apoptosis. Therefore, we suggest that lovastatin-induced protein synthesis inhibition might not contribute to the concomitant neuroblasts apoptosis previously observed.
Subject(s)
Central Nervous System/drug effects , Lovastatin/pharmacology , Neurons/drug effects , Protein Biosynthesis/drug effects , RNA Cap-Binding Proteins/antagonists & inhibitors , Animals , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/physiology , Dose-Response Relationship, Drug , Neurons/cytology , Neurons/physiology , Protein Biosynthesis/physiology , RNA Cap-Binding Proteins/biosynthesis , RNA Cap-Binding Proteins/genetics , RatsABSTRACT
The small molecule 4EGI-1 was identified as an inhibitor of cap-dependent translation initiation owing to its disruption of the eIF4E/eIF4G association through binding to eIF4E. 4EGI-1 exhibits growth-inhibitory and apoptosis-inducing activity in cancer cells; thus, we were interested in its therapeutic efficacy in human lung cancer cells. 4EGI-1, as a single agent, inhibited the growth and induced apoptosis of human lung cancer cells.When combined with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), enhanced apoptosis-induced activity was observed. As expected, 4EGI-1 inhibited eIF4E/eIF4G interaction and reduced the levels of cyclin D1 and hypoxia-inducing factor-1alpha (HIF-1alpha), both of which are regulated by a cap-dependent translation mechanism. Moreover, 4EGI-1 induced CCAAT/enhancer-binding protein homologous protein-dependent DR5 expression and ubiquitin/proteasome- mediated degradation of cellular FLICE-inhibitory protein (c-FLIP). Small interfering RNA-mediated blockade of DR5 induction or enforced expression of c-FLIP abrogated 4EGI-1's ability to enhance TRAIL-induced apoptosis, indicating that both DR5 induction and c-FLIP down-regulation contribute to enhancement of TRAIL-induced apoptosis by 4EGI-1. However, inhibition of eIF4E/eIF4G interaction by knockdown of eIF4E effectively reduced the levels of cyclin D1 and HIF-1alpha but failed to induce DR5 expression, downregulate c-FLIP levels, or augment TRAIL-induced apoptosis. These results collectively suggest that 4EGI-1 augments TRAIL-induced apoptosis through induction of DR5 and down-regulation of c-FLIP, independent of inhibition of cap-dependent protein translation.
Subject(s)
Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Nitro Compounds/pharmacology , Protein Biosynthesis/drug effects , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Thiazoles/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cells, Cultured , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Drug Synergism , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/metabolism , Humans , Hydrazones , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Nitro Compounds/administration & dosage , Protein Binding/drug effects , Protein Biosynthesis/genetics , RNA Cap-Binding Proteins/antagonists & inhibitors , RNA Cap-Binding Proteins/physiology , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , Thiazoles/administration & dosageABSTRACT
The shut-off of host protein synthesis in virus-infected cells is one of the important mechanisms for viral replication. In this report, we showed that the HL strain of measles virus (MeV-HL) as well as other field isolates, which were isolated from human blood lymphocytes using B95a cells, induce the shut-off in B95a cells. Since the Edmonston strain of MeV failed to induce the shut-off in B95a cells, the ability to induce the shut-off was considered to be dependent on virus strains. Although, the modification of eukaryotic translation initiation factors (eIF) including eIF4G, eIF4E, and 4E-BP1 was reported for shut-off by various viruses, the involvement of these eIFs was not observed in MeV-HL-infected B95a cells. Instead, the accumulation of phosphorylated eIF2alpha was found to coincide to the decrease of host protein synthesis, suggesting the involvement of phosphorylation of eIF2alpha in inhibition of translation as one of the mechanisms of the shut-off.